Cargando…
Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples
Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequenci...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7512157/ https://www.ncbi.nlm.nih.gov/pubmed/32727827 http://dx.doi.org/10.1128/JCM.00632-20 |
_version_ | 1783586097330126848 |
---|---|
author | Cabibbe, Andrea M. Spitaleri, Andrea Battaglia, Simone Colman, Rebecca E. Suresh, Anita Uplekar, Swapna Rodwell, Timothy C. Cirillo, Daniela M. |
author_facet | Cabibbe, Andrea M. Spitaleri, Andrea Battaglia, Simone Colman, Rebecca E. Suresh, Anita Uplekar, Swapna Rodwell, Timothy C. Cirillo, Daniela M. |
author_sort | Cabibbe, Andrea M. |
collection | PubMed |
description | Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION’s portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases. |
format | Online Article Text |
id | pubmed-7512157 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-75121572020-10-02 Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples Cabibbe, Andrea M. Spitaleri, Andrea Battaglia, Simone Colman, Rebecca E. Suresh, Anita Uplekar, Swapna Rodwell, Timothy C. Cirillo, Daniela M. J Clin Microbiol Mycobacteriology and Aerobic Actinomycetes Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION’s portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases. American Society for Microbiology 2020-09-22 /pmc/articles/PMC7512157/ /pubmed/32727827 http://dx.doi.org/10.1128/JCM.00632-20 Text en Copyright © 2020 Cabibbe et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Mycobacteriology and Aerobic Actinomycetes Cabibbe, Andrea M. Spitaleri, Andrea Battaglia, Simone Colman, Rebecca E. Suresh, Anita Uplekar, Swapna Rodwell, Timothy C. Cirillo, Daniela M. Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title | Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title_full | Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title_fullStr | Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title_full_unstemmed | Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title_short | Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples |
title_sort | application of targeted next-generation sequencing assay on a portable sequencing platform for culture-free detection of drug-resistant tuberculosis from clinical samples |
topic | Mycobacteriology and Aerobic Actinomycetes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7512157/ https://www.ncbi.nlm.nih.gov/pubmed/32727827 http://dx.doi.org/10.1128/JCM.00632-20 |
work_keys_str_mv | AT cabibbeandream applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT spitaleriandrea applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT battagliasimone applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT colmanrebeccae applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT sureshanita applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT uplekarswapna applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT rodwelltimothyc applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples AT cirillodanielam applicationoftargetednextgenerationsequencingassayonaportablesequencingplatformforculturefreedetectionofdrugresistanttuberculosisfromclinicalsamples |